The LHD and the Clinical Pathophysiology Section (CPS) within it, have a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulin E and recurrent infections (Job's Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. 1) A consequence of long term natural history studies of rare diseases is that with sufficiently large cohorts, statistically well-powered and unexpected findings may be made. Our studies completed and published during FY11 of 244 distinct families, indicate that survival in CGD patients was highly associated with residual reactive oxygen intermediate (ROI) production as a continuous variable, independent of the specific gene affected. Patients with mutations in p47phox and most patients with missense mutations in gp91phox (excepting mutations of the nucleotide binding and the heme binding domains) had more residual ROI production than patients with nonsense, frameshift, splice or deletion mutations in gp91phox. After adolescence, mortality curves diverged based on residual ROI production. These data have important implications for the management of patients with CGD. Although our data indicate that residual ROI production predicts mortality in CGD, genetic analysis can, in many cases, predict ROI production. In our studies of 273 fully genotyped patients 154 mutations were found, including 55 of which have not been reported previously with 45 in gp91phox, 3 in p22phox and 7 in p67phox. Missense and frameshift mutations in gp91phox were randomly distributed throughout the gene and were generally family specific. Little residual ROI production was observed with nonsense, frameshift, splice or deletion mutations in gp91phox. Unexpectedly, patients with missense mutations affecting amino acids 1-309 (excepting heme-binding His222) exhibited even higher residual ROI production than was observed in patients with p47phox CGD. ROI production was not correlated with protein expression. Mutations in FAD and NADPH binding domains of gp91phox may allow normal protein expression but little residual ROI production demonstrating and confirming the critical role of these domains. While protein detection provides important clues to the diagnosis of CGD, quantitiative measure of ROI, which correlates closely with specific genetic mutations, is more useful for determination of a patients long-term risk and thereby guides therapeutic management. In addition, the Neutrophil Monitoring Laboratory (NML), our contract clinical laboratory, has characterized the molecular defects by western blotting of 28 additional CGD patients, and by nucleic acid sequencing of 90 CGD patients or carriers. 2) Our group has been involved in the clinical and laboratory studies of the novel Gram-negative CGD pathogen, Granulibacter bethesdensis. During FY11, we have purified the dominant antigen recognized by patient sera, methanol dehydrogenase (MDH), and have begun the biochemical characterization of this enzyme. In collaboration with the LCID, we have participated in serologic studies of the CGD cohort using both immunoblotting and an ELISA based on MDH. These studies have identified several additional CGD patients with evidence of likely prior infections with Granulibacter and provide tools for larger-scale screening of other patient population in which this infection may be suspected. As yet unpublished transcriptional analysis of Granulibacter exposed to normal and CGD neutrophils suggests that this enzyme, upregulated as it is within the neutrophil, may play a role during infections. Upcoming biochemical studies of the native enzyme may provide insight into its role during infection and, perhaps, provide impetus to develop specific inhibitors to use as targeted antimicrobial therapy. 3) During FY2011, we have continued the enrollment of patients into NIH Protocol #10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System. Atherosclerosis, the major cause of heart disease, is thought to relate to dysregulated inflammation in the cardiac blood vessels and possibly results from over production of reactive oxygen species (ROS). We hypothesize that CGD patients, who have deficient production of reactive oxygen species by their phagocytes, may be protected from developing atherosclerosis. The primary endpoint of this study is the assessment of atherosclerotic plaque formation/calcium deposition by CT, MRI and other imaging methodologies, in these and other patients with in-born disorders of immune function. Inflammatory bowel disease provides a group of patients with similar chronic inflammation but with intact ROS production for comparison. Importantly, this study may determine for the first time in humans, whether ROS indeed play a role in the pathogenesis of atherosclerosis, a finding that could have broad importance for the general population as it would provide a validated molecular target for therapeutic intervention. Given the potential role of the NADPH oxidase in the regulation of inflammation in atherosclerosis and possibly other inflammatory diseases, we have recently initiated a project in collaboration with the National Center for Genomic Chemistry (NCGC/NHGRI). They have synthesized and provided compounds which we have tested in whole cell Nox2 assays (luminol-enhanced neutrophil chemiluminscence) resulting in the identification of several interesting compounds (both inhibitors and activators of Nox2). Whether such inhibitors might represent useful drugs in the treatment of cardiovascular disease or whether activators may be able to boost ROS production in autosomal CGD will be tested in the next year.